Ayuso's People's Party (PP) has governed Spain's wealthiest region in coalition with Aguado's centrist Ciudadanos since 2019, but long-standing tension between the two parties boiled over on Wednesday, threatening several jointly controlled regional authorities.
The snap election will be held on May 4.
Aguado criticised Ayuso's sudden resignation and said he hoped she would reconsider.
"It seems terribly reckless to us to call elections ... and open the door to instability that could cause the Madrid region to derail from a health, economic and social point of view," he told reporters.
Madrid's regional administration is in charge of health policy and is responsible for its own coronavirus response and vaccine rollout.
In southeastern Murcia, where the two parties also have a coalition, Ciudadanos sided with Prime Minister Pedro Sanchez's Socialists in bringing a no-confidence motion against regional leader Fernando Lopez Miras of the PP.
By publicly breaking ranks with the PP, Ciudadanos has raised the spectre of similar no-confidence votes in other regions where they rule in coalition, such as Andalusia and Castille and Leon.
For now, Ciudadanos in Andalusia - Spain's most populous region - have said their pact is stable, while in Castille and Leon, both parties said it remains valid.
Widely viewed as a maverick, Ayuso has frequently clashed with the left-wing central government. Her administration imposed one of Spain's loosest curfews and has consistently refused to shut bars and restaurants, in defiance of national recommendations.
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